Method for controlling sample introduction in microcolumn separation techniques and sampling device

ABSTRACT

In a method for controlling sample introduction in microcolumn separation techniques, more particularly in capillary electrophoresis (CE), where a sample is injected as a sample plug into a sampling device which comprises at least a channel for the electrolyte buffer and a supply and drain channel for the sample. The supply and drain channels discharge into the electrolyte channel at respective supply and drain ports. The distance between the supply port and the drain port geometrically defines a sample volume. The injection of the sample plug into the electrolyte channel is accomplished electrokinetically by applying an electric field across the supply and drain channels for a time at least long enough that the sample component having the lowest electrophoretic mobility is contained within the geometrically defined volume. The supply and drain channels each are inclined to the electrolyte channel. Means are provided for electrokinetically injecting the sample into the sample volume. The resistance to flow of the source and drain channels with respect to the electrolyte buffer is at least about 5% lower than the respective resistance to flow of the electrolyte channel.

This application is a continuation of application Ser. No. 08/226,605,filed Apr. 12, 1994, now U.S. Pat. No. 6,280,589 which is incorporatedherein by reference in its entirety.

The present invention concerns a method for controlling sampleintroduction in microcolumn separation techniques. The invention alsoconcerns a respective sampling device for a controlled sampleintroduction in microcolumn separation techniques.

BACKGROUND OF THE INVENTION

Microcolumn separation techniques, in particular capillaryelectrophoresis has become a very interesting separation technique whichis used as part of a sensor or a chemical analysis system. One majorreason for this is the great efficiency of the method as a separationtechnique. The sampling methods usually applied in capillaryelectrophoresis are: injection of a sample with a syringe, via a septum,in an injection block, the use of injection valves with/without a sampleloop, and dipping one end of the capillary tube into the samplereservoir, whereby the sample is introduced by gravity flow, by over- orunderpressure, or by electroendosmosis and/or electromigration.

While it is mentioned in Journal of Chromatography, 452, (1988) 612-622,that sample valves are the most suitable sampling method for capillaryelectrophoresis, there also is described a valveless device for theinjection of a sample. The described arrangement comprises a castcapillary block which is connected between an electrode compartment anda sampling device. In the electrode compartment electrolyte solutionscontact electrodes. The capillary tube contains measuring electrodeswhich are connected with an evaluation electronics. The sampling deviceconsists of a broadened part of the capillary tube connected with twofeeders which extend perpendicular to the capillary tube. Thearrangement of the two feeders off-set from each other along thelongitudinal extension of the capillary tube is such, that the samplingdevice has the shape of a capillary double T structure.

The sample is introduced into the sampling device via a syringe. Theinjection volume is defined geometrically by the distance which the twofeeders are spaced apart along the capillary tube. The transport of theelectrolyte solution and the sample in the capillary tube isaccomplished by electric fields that are applied between the respectiveelectrodes along the capillary tube. An advantage of the double T shapesampling device, as is also obtained with the use of injection valves,is the concentration effect of dilute sample ionic species. However, itis possible that, allthough no electric field gradient over the feedersexists, sample components from the feeders may diffuse into thecapillary tube when the sample has already left the sampling position.The amounts of sample components that uncontrollably enter the capillarytube depend on the diffusion coefficients and the mobilities of therespective sample components. Thus, at the detector there not onlyarrives a more or less broadened plug of injected sample fluid,depending on the diffusion coefficients and the mobilities of therespective components in the electrolyte and the electric field, butalso the electrolyte in front and after or between individual plugs ofsample fluid is “polluted” with unpredictable amounts of samplecomponents. These unpredictable amounts of sample components reachingthe detector are highly undesirable and result in a high noise of thedetected signal, thus reducing the limits of detection considerably.

In Analytical Chemistry, 1992, 64, pages 1926-1932 a capillaryelectrophoretic device is described in which the sample is injectedelectrokinetically dipping one end of a capillary into the samplereservoir and applying a voltage across the ends of the capillary. Inthe electric field the sample is transported electrokinetically and isinjected at a T-junction into the channel system of the capillaryelectrophoretic device. This method, however, leads to a well-known biasof the actual sample composition due to the differences in theelectrophoretic mobilities of the sample components. Thus, the sampleintroduced often does not have the same composition as the originalsample. In addition, the volume of the introduced sample is very oftenunknown such, that internal standards have to be used for quantitativeanalyses.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a methodfor controlling sample introduction in microcolumn separationtechniques, and more particularly in capillary electrophoresis (CE), anda sampling device which overcomes the aforementioned disadvantages ofthe prior art. The sample volume shall be geometrically defined. Thecomposition of the sample which is injected shall not differ from theoriginal composition of the sample in the reservoir. The uncontrolledintroduction of sample fluid into the capillary tube shall be reducedconsiderably. If the unwanted leakage of sample fluid into the capillarytube cannot be totally avoided, provisions shall be made that at leastit only occurs in a predictable and controllable manner.

The method and the sampling device according to the invention shall alsoallow an easy realization of miniaturized analysis concepts, such as theones described, for example, in Sensors and Actuators B, 10 (1993)107-116. There the concept of a multi-manifold flow system integrated ona silicon substrate, with valveless switching of solvent flow betweenchannels and electro-kinetic pumping of an aqueous solvent, isdescribed. A similar concept is described, for example, in AnalyticalChemistry , Vol.64, No. 17, Sep. 1, 1992, 1926-1932. The describedminiaturized chemical analysis system on the basis of capillaryelectrophoresis comprises a complex manifold of capillary channels,which are micromachined in a planar glass substrate. The transport ofthe solvent and the sample occurs due to electro-kinetic effects(electro-osmosis and/or electrophoresis).

In order to meet all these and still further objects according to theinvention a method for controlling sample introduction in microcolumnseparation techniques, in especially in capillary electrophoresis (CE)is provided, wherein an electrolyte buffer and a more or lessconcentrated sample are transported through a system of capillarychannels. The sample is injected as a sample plug into a sampling devicewhich comprises at least a channel for the electrolyte buffer and asupply and drain channel for the sample. The channel for the electrolytebuffer and the supply and drain channels for the sample intersect eachother. The supply channel and the drain channel for the sample, eachdischarge into the channel at respective supply and drain ports. Thedistance between the supply port and the drain port geometricallydefines a sample volume. The supply and the drain channels each areinclined to the electrolyte channel. The injection of the sample pluginto the electrolyte channel is accomplished electro-kinetically byapplying an electric field across the supply and drain channels for atime at least long enough that the sample component having the lowestelectrophoretic mobility is contained within the geometrically definedvolume. By this measure the composition of the injected sample plug willreflect the actual sample composition.

In a further preferred process step, immediately after the injection ofthe sample plug, the electrolyte buffer is allowed to advance into thesupply channel and into the drain channel at the respective supply anddrain ports for a time period, which amounts to at least the migrationtime of a slowest component within the sample plug from the supply portto the detector. Thus, the sample is pushed back into the respectivesupply and drain channels and substantially prevented fromuncontrollably diffusing into the electrolyte buffer which istransported past the supply and drain ports. In addition the methodallows to control the sample composition within the electrolyte buffer.

The sampling device according to the invention comprises an electrolytechannel, and a supply channel and a drain channel for the sample, whichdischarge into the electrolyte channel at respective supply and drainports. The ports are arranged with respect to each other such, that asample volume is geometrically defined. The supply and drain channelseach are inclined to the electrolyte channel. Means are provided forelectro-kinetic ally injecting a sample into the sample volume. Theresistance to flow of the source and drain channels with respect to theelectrolyte buffer is at least about 5% lower than the respectiveresistance to flow of the electrolyte channel. Preferred variants of themethod according to the invention and preferred embodiments of thesampling device according to the invention are subject of the respectivedependent claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will become apparent from the following description withreference to the schematic drawings in which:

FIG. 1 is a schematic view of a microcolumn separation device whichcomprises a sampling device according to the present invention,

FIG. 2 is a sectional view of the microcolumn separation deviceaccording to FIG. 1,

FIG. 3 is an enlarged view of the encircled part of the microcolumnseparation device according to FIG. 1, showing a first embodiment of asampling device, and

FIG. 4 is a second embodiment of the sampling device.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In FIGS. 1 and 2 an exemplary embodiment of a microcolumn separationdevice, more particularly of an electrophoretic separation device, isdepicted. It comprises a base part 1 and a lid part 2. The base part 1can be made of glass, monocrystalin silicon or other materials knownfrom semiconductor manufacture, or of a suitable polymer material. Thelid part 2 is preferably made of glass. The base part 1 comprises achannel system 4 which is etched, micromachined or otherwise establishedin its surface. Preferably techniques known from semiconductormanufacture are applied for creating the channel system in the surfaceof the base part 1. The lid part is provided with through holes R, S, D,W, which communicate with the channel system 4 and are adapted toaccomodate and hold the ends of capillary tubes. The lid part 2 is alsoprovided with various ports for light waveguides, which are part of anoptical detection system, such as, for example, a fluorescence detectionsystem, or an absorption detection system, or a system for the detectionof changes of the refractive index of a sample flowing through thechannel system. The ports are distributed along the channel system 4after a sampling device 3, where a sample is introduced into anelectrolyte buffer, thus allowing measurements at different locationsalong the channel system.

The transport of the electrolyte buffer and of the more or lessconcentrated sample is preferably accomplished by means of electricfields, which are created by switching electric potentials betweenelectrodes of a respective reservoir R and waste receptacles W for theelectrolyte buffer and between electrodes associated with respectivesource S and drain receptacles D for the sample.

In FIGS. 3 and 4 the encircled sampling device 3 of FIG. 1 is shown inan enlarged scale. It is part of the flow injection analysis system ofFIG. 1, which is based on electro-kinetic principles and allows anelectrophoretic analysis of a sample. The sampling device 3 is anintegrated part of the capillary channel system 4 and is thus connectedwith the reservoir R and the waste receptacle W behind the detectors5-8, for the electrolyte buffer, and with the source receptacle S andthe drain receptacle D for the sample which is to be analyzed. In FIGS.3 and 4, for the sake of clarity the reservoir R and the receptacles W,S, D are not drawn, but they are only symbolized by arrows, which at thesame time indicate the direction of fluid flow in the channel system 4.

In FIG. 3 a first exemplary embodiment of the sampling device is shown.It comprises a capillary channel piece 22, which on one end is connectedto a capillary channel communicating with the reservoir R for theelectrolyte buffer and in longitudinal direction on the other end with acapillary channel where the electrophoretic separation of the sampletakes place, and which leads to the detector(s) and in furtherconsequence to the waste receptacle(s) W. The sampling device furthercomprises a supply channel 23, which communicates with a sourcereceptacle S for the sample, and a drain channel 24 which leads to adrain receptacle D. The source channel 23 and the drain channel 24 areinclined to the longitudinal extension of the channel piece 2,preferably they are arranged about perpendicular such, that togetherwith the channel piece 22 they form a double T structure, as shown inthe drawing. The source channel S and the drain channel D each dischargeinto the channel piece 22 at respective supply and drain ports 25, 26.According to the drawing in FIG. 3 the supply port 25 and the drain port26 are spaced apart from each other longitudinally at the channel piece22 such, that a sample volume 27 is geometrically defined as will beexplained in more detail hereinafter. It is to be understood, that thedrain channel 24 can be arranged in direct longitudinal extension of thesource channel 23 such, that the supply and drain ports 25, 26 aresituated opposite each other. In that case the channels of the samplingdevice have no double T structure, but they are arranged in form of anordinary crossing.

As already mentioned before the transport of the fluids, i.e. theelectrolyte buffer and the sample, is accomplished with electric fields,which are a result of different electric potentials at the reservoir Rand the waste receptacle W for the electrolyte buffer, and therespective source receptacle S and the drain receptacle D for thesample. By applying, for example, a positive electric potential to thereservoir R and a negative electric potential to the waste receptacle,the electrolyte buffer is electro-kinetically transported from thereservoir R through the capillary channel system to the waste receptacleW. In order to introduce the sample into the channel piece 22, forexample, the source receptacle S for the sample is maintained at apositive potential and the drain receptacle D is kept on a negativepotential. In the resulting electric field the sample is transportedelectro-kinetically from the source receptacle S to the drain receptacleD. The direction of flow is indicated in FIG. 3 by the arrows S, V, andD. By this measure, a part 27 of the channel piece 22, which isdelimited by the supply port 25 on the one end and by the drain port 26on the other end is filled with sample. Thus, the sample-filled part 27of the channel piece of the sampling device 3 defines the volume of theelectro-kinetically injected sample plug, which is indicated by thehatchings in FIG. 3. In other words, the volume 27 of the sample plug isgeometrically delimited by the spaced apart supply and drain ports 25and 26. In the aforemantioned case that the supply and drain ports arearranged opposite each other, such that the channel piece 22 and thesupply and drain channels 23, 24 form an ordinary crossing, the size andvolume of the intersection determines the sample volume. Thus, in thatcase, the sample volume is only defined by the cross-sections of therespective channels 22, 23, 24.

In order to assure that the composition of the sample in the samplevolume 27 reflects the actual sample composition in the reservoir R theelectric field across the supply and drain channels 23, 24 must bemaintained for at least for a time period long enough that thegeometrically defined sample volume is filled and contains the thecomponent of the sample which has the lowest electrophoretic mobility.This minimum time period t_(min) is given by the equationt_(min)=d/μ_(i)·E. In this equation d stands for the distance, which thesource and drain port are spaced apart; μ_(i) is the total mobility ofthe slowest component i of the sample, which will be referred to in moredetail hereinafter, E is the field strength across the source and drainchannels, which results from the difference in potentials.

When a electrophoretic analysis of a sample is to be carried out, firstan electric field between the reservoir R and the waste receptacle W isestablished such, that the electrolyte buffer is transported from thereservoir R to the waste receptacle W. After the channel system of thechemical analysis system has been filled with the electrolyte buffer,the injection of the sample into the channel piece 22 is initiated. Forthat purpose an electric field is established between the sourcereceptacle S and the drain receptacle D such, that the sample iselectro-kinetically transported from the source receptacle S through thesupply channel 23 via the channel piece 22 into the drain channel 24 andon to the drain receptacle D. It is understood that during the timeperiod, in which the sample is injected, the electric field between thereservoir R and the waste receptacle W is switched off, or that thepotentials are chosen such, that the sample only is transported alongthe path described above. After the injection time period which ischosen such, that it is ensured that the sample volume 27 between thesupply port 25 and the drain port 26 is filled with the sample, theelectric field between the source receptacle S and the drain receptacleD is switched off. At the same time the electric field between thereservoir R and the waste receptacle W is activated again such, that thesample contained within the sample volume 27 is transported on into thedirection of the detector(s) and the waste reservoir. While it istransported through the channel system the sample is separatedelectrophoretically in the electric field.

The problem of leakage or diffusion of sample components into theelectrolyte buffer while it is transported past the supply and drainports 23 and 24, even though no electric field is applied between thesource receptacle S and the drain receptacle D, is solved by allowingthe electrolyte buffer to advance into the supply channel 23 and intothe drain channel 24 at the respective supply and drain ports 25 and 26for a time period, which amounts to at least the migration time t_(i) ofthe slowest component i within the sample plug from the supply port 25to the respective detector. Thus, the sample is pushed back into thesupply and drain channels 23, 24 and substantially prevented fromuncontrollably diffusing into the electrolyte buffer which istransported past the supply and drain ports 25, 26.

The migration time t_(i) of the slowest component i of the sample isdefined as the quotient between the separation length L and the productof the total mobility μ_(i) of the slowest component i of the sample andthe electric field strength E′ acting on it along its path L, and isgiven by the equation T_(i)=L/(μ_(i)·E′). In this equation theseparation length L (FIG. 1) is the distance the sample component itravels between the supply port 25 and the respective activated detector5-8, and the total mobility μ_(i) of the component is the sum of theelectrophoretic mobility μ_(i,ep) of the component and the overallelectro-osmotic mobility μ_(eo) of the sample. The time period duringwhich the detection is accomplished is very short in comparison to themigration time of the slowest component of the sample and thus isneglectable.

In order to allow the electrolyte buffer to advance into the supply anddrain channels 23 and 24, in the exemplary embodiment of the samplingdevice depicted in FIG. 3 the source receptacle S and the drainreceptacle D are switched on an electric potential which is differentfrom the electric potential at the reservoir R for the electrolytebuffer, thus establishing a potential difference of suitable magnitude.In an embodiment, where the electrolyte buffer is transported from apositive potential to a negative potential, the potentials at the sourceand drain receptacles S, D are chosen negative with respect to thepositive potential at the reservoir R. In case of a transport of theelectrolyte buffer from a negative potential to a positive potential thepotentials of the source and drain receptacles S, D are chosen positivewith respect to the reservoir R.

Preferably the potential difference between the reservoir R and thesource and drain receptacles S, D is chosen such, that the resultantelectric field has a field strength which amounts to at least about 0.1V/cm.

Another approach to allow an advancement of the electrolyte buffer intothe supply and drain channels 3, 4 is depicted in FIG. 4. Theconstruction of the depicted sampling device 3 basically corresponds tothe one depicted in FIG. 3. It comprises a channel piece 12 with twoside channels 13, 14. The side channels are inclined to the longitudinalextension of the channel piece 12 an angle that amounts to from about 5degrees to about 175 degrees; however, preferably they are arrangedabout perpendicular with respect to the channel piece 12. The sidechannels are a supply channel 13 and a drain channel 14, which dischargeinto the channel piece 12 at respective supply and drain ports 15, 16.Preferably the supply port 15 and the drain port 16 are spaced apartfrom each other at the channel piece 12 and delimit a sample volume 17.The distance d which they are spaced apart from each other typicallyamounts to from about 0 μm to about 3 cm, most preferably to about 3 mm,wherein the value 0 indicates that the supply and drain ports arelocated opposite each other. The channel piece 12 communicates with areservoir R and a waste receptacle W for the electrolyte buffer. Thesupply channel 13 is connected with a source receptacle S for thesample, while the drain channel 14 communicates with a drain receptacleD.

The sampling device 3 is part of an electrophoretic chemical anlysissystem and basically functions in the same way as the sampling devicedepicted in FIG. 3. However, in order to allow the electrolyte buffer toadvance into the supply and drain channels 13, 14 the resistance of flowwithin the two channels is reduced. In particular the source channel andthe drain channel each have a resistance to flow with respect to saidelectrolyte buffer, which is about 5% lower than the respectiveresistance to flow of said electrolyte channel. Surprisingly thereduction of the resistance to flow of the supply and drain channels 13,14 does not result in an increase of the leakage or diffusion of samplecomponents into the electrolyte buffer as it is transported past therespective supply and drain port 15, 16. Instead, the reduction of theresistance to flow of the side channels 13, 14 leads to a convectiveflow of the electrolyte buffer into the channels 13, 14, even when theaplied electric fields should not result in such a flow. Thus, theleakage or diffusion of sample components is considerably decreased andthe noise of the detected signal is reduced. In consequence thesensitivity of the analytic system, that is the limit of detection, isincreased. The resistance to flow of the supply and drain channel can bedeminished by either reducing the length of the respective channels orby increasing their respective widths w. Preferably the reduction of theresistance to flow of the supply and drain channels 13, 14 is achievedby providing them each with a width w that is at least about two timesgreater than the width p of the supply and drain ports 15, 16. Such, thesupply and drain channels 13, 14 each have about the shape of a bottle,the bottle neck being the respective supply or drain port 15, 16.

While it is possible that the supply and drain channels 13, 14 emptydirectly into the channel piece 12 such, that their ends, which arelocated right next to the channels piece 12 are the respective sourceand drain ports 15, 16, from where the width of the channels graduallyincreases over a respective intermediate piece 13′, 14′ from the width pof the ports to the final width w of the channels, the supply and drainports also have longitudinal extensions l. Thes longitudinal extensionscorrespond at least to the width p of the respective supply and drainports 15, 16. It is advantageous, if the widths p of the supply anddrain port 15, 16 are kept constant along their longitudinal extensionl. In a preferred embodiment the widths p of the supply and drain port15, 16 are chosen such, that they about correspond to the width b of thechannel piece 12.

The depth of the channel piece 12 (which should correspond to the depthof the channel system that it is part of) and of the supply and drainchannels 13, 14 typically amounts to from about 0.1 μm to about 100 μm.The depths of the bottle-neck-like supply and drain port 15, 16 aboutcorresponds to the depth of the channels.

The sampling device 3 according to the invention has been explained withreference to exemplary embodiments which are part of micro-analysischips. It can as well be an arrangement of capillary tubes, which ispart of a electrophoretic chemical analysis system made of capillarytubes. In the most preferred embodiment, however, the sampling device isintegrated into a system of capillary channels which are established ina small planar sheet of glass, semiconductor material, or a suitablepolymer. Advantageously the channel system including the supply anddrain channels and the respective supply and drain ports are etched ormicromachined or casted (in case of a polymer base part), or otherwiseestablished in the planar substrate. Most suitable for its manufactureare techniques which are well established in semiconductor production orin the manufacture of micromechanical elements.

The combination of a structure that geometrically defines the injectedsample volume with an electro-kinetic injection of the sample over adefined minimum time period allows to relyably control the sample volumeand to assure that the composition of the sample contained within thesample volume reflects the original composition of the sample in thereservoir. A further improvement of the method and the sampling deviceaccording to the invention allows a considerable reduction ofuncontrolled leakage or diffusion of sample components into theelectrolyte buffer. Thus, it is possible to reduce the leakage ordiffusion such, that the still occuring leakage results in aconcentration of the sample in the electrolyte buffer, that is less than3% of the original concentration of the sample. By this measure thenoise of the detected electrophoretic signal is reduced and thedetection limits are increased.

What is claimed is:
 1. A microfluidics device comprising a substrate having an upper surface, a channel network formed in the substrate, including a source channel adapted to contain a sample, a drain channel, and an electrolyte channel adapted to contain an electrolyte buffer, wherein said source and drain channels are each inclined with respect to the electrolyte channel, and which source and drain channels intersect said electrolyte channel at a source port and a drain port, respectively, wherein movement of sample material from the source to the drain channel is effective to place a sample volume between the source and drain ports in the electrolyte channel, a lid attached to the substrate's upper surface, enclosing the channels in the substrate, and openings in the lid communicating with the channel network and adapted to hold capillary tubes therein, for introducing liquid from the tubes into associated channels in the device.
 2. The device of claim 1, wherein said channels terminate at and are in fluid communication with reservoirs, and said openings are aligned to communicate with said reservoirs.
 3. The device of claim 1, wherein said source and drain ports are offset along the length of said electrolyte channel, forming an elongate sample volume within said electrolyte channel, between and including said ports. 